The overall goal of this grant is to determine the mechanism by which the ATP-binding cassette halftransporters, ABCG5 and ABCG8 (G5G8), mediate the transport of sterols across membranes. Previously, we discovered that mutations in either G5 or G8 cause sitosterolemia, which is characterized by the accumulation of plant sterols and cholesterol, and by premature coronary artery disease. We showed that G5 and G8 must heterodimerize in the endoplasmic reticulum (ER) prior to trafficking to the apical surfaces of hepatocytes and enterocytes, where the transporter secretes sterols into bile and the gut lumen, respectively. During the last funding period we developed in vivo and in vitro assays to assess the structural requirements for G5G8- mediated sterol transport.